The present invention broadly relates to methods for detecting binding interactions between proteins and various types of ligands. More specifically, the present invention relates to methods for screening large collections of ligands for those having specific affinity for a protein target of interest. As such, the present invention is useful within the fields of fundamental biomedical and biochemical research, especially drug discovery and medical diagnostics.
Proteins play a variety of key roles in biological processes and functions, including for example, functioning as catalysts, regulators of biochemical pathways, receptors, and as important elements in immune response. Given their diverse and important roles, it is not surprising that ligands that bind to proteins have been viewed by pharmaceutical researchers as attractive candidates for therapeutic agents. One traditional approach for drug discovery simply involved making modifications to natural regulators. As more data regarding structure function relationships became available, it became possible to engage in rational drug design using computers and x-ray structures to aid in synthesizing molecules tailored to fit the active site of an enzyme, for example. However, even using such advanced techniques, drug screening and development remained an often tedious and time consuming process.
More recent drug discovery methods take a different approach and involve screening extremely large libraries of compounds for their ability to bind protein targets of interest. This type of approach typically begins with the identification of a potential protein target, such as a receptor for example. A diverse library is then prepared containing ligands to be screened for their ability to bind the target. The libraries may be random peptide libraries, carbohydrate libraries, natural product libraries, etc. Often the libraries are prepared using recently developed combinatorial techniques. These libraries are subsequently subjected to high throughput screening to identify ligands that bind to the target. Because the key feature of this approach is to screen a huge number of molecules, the success of this approach hinges on the ability to rapidly screen and identify ligands that do bind the target. Ligands initially identified as binding the target are then used to develop more focused libraries that are then put through the same screening process. This process of screening and preparing new focused libraries typically is repeated several times until a relatively small population of lead compounds are identified. These lead compounds are then subjected to various pharmaceutical analyses to select useful drug candidates.
A primary limitation in current methods is that the screening tests simply detect binding, but are unable to distinguish between specific and non-specific binding. Some approaches also are not fully compatible with high-throughput screening procedures. Moreover, many current screening methods require labeling of either the target or ligand and are unable to detect binding complexes directly.
The present invention provides novel methods for analyzing protein binding events in which the formation of protein/ligand complexes can be directly detected. Using this system, it is possible to screen libraries on the basis of specific binding interactions. It is also possible to perform a variety of analytical and diagnostic analyses with the system of the present invention.
The present invention generally provides methods for detecting binding events between proteins and a variety of different types of ligands utilizing a system which is sensitive to the dielectric properties of molecules and binding complexes such as protein/ligand complexes. Other methods involve screening libraries of ligands to identify those ligands which bind to a protein of interest, such methods have particular utility in drug screening programs, for example. Other methods are diagnostic methods in which the system is used to detect the presence of a particular ligand that binds to a known protein, or of a particular protein that binds to a known ligand. The screening and diagnostic methods can be performed using arrays having multiple elements.
More specifically; some methods involve obtaining a spectrum for a protein/ligand complex. Such methods include acquiring a spectrum for a protein/ligand complex formed between a protein and a test ligand. The spectrum is acquired by propagating a test signal along a signal path and detecting a response signal for the protein/ligand complex, wherein the protein or test ligand is electromagnetically coupled to a portion of the signal path. The test signal propagated along the signal path is varied with time to obtain the spectrum. The test signal is varied, for example, by altering the frequency or wavelength with time.
Certain methods involve screening ligands for the ability to bind to a target protein or protein of interest. The method includes contacting a protein of interest with a ligand. The formation of a protein/ligand complex is detected through the formation of a response signal resulting from the complex. Typically, either the protein of interest or test ligand is electromagnetically coupled to a portion of a continuous transmission line.
Some screening methods of the present invention are more sophisticated and include acquiring a spectrum for a protein/ligand complex between a known protein and a test ligand wherein either the known protein or test ligand are electromagnetically coupled to a portion of a signal path. The spectrum is acquired by propagating a test signal that is varied with time along the signal path and detecting a response signal for the complex between the known protein and the test ligand. The resulting spectrum is then examined for the presence of a known signal which is characteristic for the binding of a known ligand at a particular site on the known protein. The presence of the known signal in the spectrum is indicative of the test ligand binding at the particular site to which the known ligand binds. For tests in which the known protein is an enzyme, the particular site can be the active site or an allosteric site, for example. When the known protein is a receptor, the particular site can be the site at which a natural ligand binds. The particular site for tests conducted with a known antibody typically is the antigen binding site for a known antigen.
In related screening methods, the spectrum is examined for the presence of a known signal which is characteristic for the binding of a particular class of ligand to the known protein. Thus, for methods in which the known protein is an enzyme, the known signal is for a complex with a competitive inhibitor or an allosteric inhibitor, for example. In instances in which the known protein is a receptor, the known signal is for a complex with an agonist or antagonist, for example.
The present invention also provides various diagnostic methods for detecting the presence of a particular protein or ligand in a sample. Hence, some methods include contacting a known protein that is electromagnetically coupled to a portion of a continuous transmission line with a sample potentially containing a particular ligand that specifically binds with the known protein. A sufficient period of time is allowed to elapse to permit the known protein and the particular ligand of interest, if present, to form a binding complex. Detection of a response signal for the binding complex is indicative of the presence of the particular ligand in the sample. Alternatively, a known ligand can be electromagnetically coupled to a portion of a transmission line and then contacted with a sample potentially containing a particular protein that forms a binding complex with the known ligand.
As with the more sophisticated screening methods, certain diagnostic methods include the use of characteristic signals for detection of the presence of a particular protein or ligand in a sample. More specifically, such diagnostic methods include contacting a known protein coupled to a portion of a signal path with a sample potentially containing a particular ligand that forms a binding complex with the known protein. A test spectrum is acquired by propagating a test signal along the signal path and detecting a response signal for the binding complex, wherein the propagating step comprises varying the test signal with time. The test spectrum is then examined for the presence of a known signal that is characteristic for the binding complex; the presence of such a signal indicates the presence of the particular ligand in the sample. Alternatively, a known ligand rather than a known protein is coupled to the signal path. In this instance, the methods include examining the acquired test spectrum for the presence of signals which are characteristic for a binding complex formed between the known ligand and a particular protein, the presence of such signals being indicative of the presence of the particular protein in the test sample.
Still other methods include the use of arrays that contain a plurality of sites or elements. Each element includes a continuous transmission line and a known protein (or plurality of proteins) electromagnetically coupled to a portion of the continuous transmission line located within the element. These elements are contacted with a sample containing a ligand. A response signal for the binding complex formed between the known protein and ligand is detected and indicates that the ligand is able to bind to the proteins. In other methods, known ligands rather than known proteins are attached to each site of the array and contacted with proteins contained in a sample.
Because the present methods involve direct detection of binding events, it is not necessary to use labeled proteins or ligands, thus simplifying the methods and reducing costs relative to other approaches for monitoring protein/ligand binding events. The ability to differentiate between different types of binding also makes it possible to much more rapidly screen for molecules that are of potential therapeutic value.